This is a renewal application of our grant DA-00564-23, which was committed to study the neurochemical mechanism of narcotic tolerance since its inception. With the recent success in the cloning of opioid receptors by others and our own work on elucidating the genomic structure of the opioid receptor gene, as well as our success in generating receptor reagents (e.g., receptor specific antibodies), we can now test the hypothesis, both in vitro and in vivo, that alteration in mu-opioid receptor activities (including receptor mediated signal transduction) may be the basis for morphine tolerance. We propose to use two approaches: Proposal 1) To first establish specific in vitro cell line models in which cloned mu- or mu- and delta-receptors are stably expressed. Then to use these models to define the molecular details involved in chronic morphine treatment induced alteration of mu-opioid receptor activities (including receptor number, properties and signal transductions) and their relationship to receptor desensitization and/or down regulation; and to determine the receptor domain(s) or possible other membrane factor(s) that are involved in this process. We will also test whether tolerance development is related to chronic morphine treatment induced covalent modifications (e.g., phosphorylation or others) of the receptor and its subsequent upcoupling from the respective G-proteins. Receptor phosphorylation and its role in receptor desensitization will be studied with various kinases activators and inhibitors and site-directed mutagenesis. To test if any alteration in receptor may be related to the mechanism of morphine tolerance we plan to perform reconstitution studies with immunoaffinity purified receptors and recombinant G-proteins. Proposal 2) In vivo approach, by using receptor mutant animal models. To generate mutant mice with receptor gene specifically disrupted (or altered) by gene-targeting. Molecular details about the relationship between mu-receptor activity and morphine addiction, as defined in Proposal I, can now be established in vivo. For the purpose of simplicity and more ease for the reviewer in reading the grant, we will present these two approaches separately as Proposal I (in vitro) and Proposal II (in vivo).